A trypsin, its encoding gene and use thereof

Abstract

The application discloses a trypsin, a coding gene and application thereof, and relates to the technical field of biology. The trypsin is any one of (1)-(3): (1) a Try3 protein, wherein the amino acid sequence is shown as SEQ ID NO. 2; (2) a protein with more than 90% identity with the Try3 protein, which is obtained by substitution, deletion and / or addition of one or more amino acid residues to the amino acid sequence of the Try3 protein; and (3) a fusion protein obtained by connecting a protein tag to the N terminal or / and C terminal of (1) or (2). The trypsin provided by the application has a wide pH adaptation range, good thermal stability, strong tolerance to various metal ions, and can well meet the needs of industrial production.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a trypsin, its encoding gene, and its applications. Background Technology

[0002] Trypsin is a proteolytic enzyme secreted by the pancreas. It plays a crucial role in the animal digestive tract, primarily participating in the digestion and breakdown of proteins in the small intestine. When food enters the small intestine, trypsin is converted from its precursor, trypsinogen, under the activation of intestinal peptidases. It specifically cleaves the peptide bonds on the carboxyl side of basic amino acids (such as lysine and arginine) in protein molecules, breaking down complex proteins into smaller peptides. These peptides are then further broken down into amino acids by other enzymes, thus providing the animal body with the raw materials needed to synthesize its own proteins and carry out other physiological activities, ensuring the smooth functioning of a series of life activities such as normal growth, development, and metabolism.

[0003] Trypsin is a serine protease with the ability to specifically hydrolyze the peptide bonds on the carboxyl side of basic amino acids (such as lysine and arginine) in proteins. In the food industry, trypsin can be used for meat tenderization, the production of hypoallergenic foods, and the development of bioactive peptides. In leather production, it is commonly used in soaking and dehairing processes, enabling the raw hide to absorb water evenly, removing interfibrous stroma, promoting the penetration of alkali and lime, and causing the leather to swell evenly. Simultaneously, it effectively hydrolyzes proteins at the hair root during dehairing, maintaining the integrity of the leather. In feed production, trypsin improves the utilization rate of proteins in feed by hydrolyzing them, making it particularly suitable for feed production for young animals. Therefore, the development of new trypsin inhibitors is of great significance to the paper, food, and feed industries. Summary of the Invention

[0004] The purpose of this invention is to provide a trypsin, its encoding gene, and its applications to address the problems existing in the prior art. This trypsin has a wide pH adaptability range and good thermal stability, which can well meet the needs of industrial production.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides a trypsin, wherein the trypsin is any one of (1)-(3):

[0007] (1) Try3 protein, the amino acid sequence of which is shown in SEQ ID NO.2;

[0008] (2) The amino acid sequence of the Try3 protein is replaced, deleted and / or added by one or more amino acid residues to obtain a protein that has more than 90% identity with the Try3 protein.

[0009] (3) The fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of (1) or (2).

[0010] The present invention also provides a gene encoding the above-mentioned trypsin.

[0011] Furthermore, the nucleotide sequence of the encoding gene is shown in SEQ ID NO.2.

[0012] The present invention also provides a gene expression cassette, comprising the above-described encoding gene.

[0013] The present invention also provides a recombinant expression vector comprising the gene expression cassette described above.

[0014] The present invention also provides a recombinant host cell comprising the above-described recombinant expression vector.

[0015] Furthermore, the recombinant host cell is a recombinant yeast.

[0016] The present invention also provides the use of the above-mentioned coding gene, gene expression cassette, recombinant expression vector or recombinant host cell in the preparation of the above-mentioned trypsin.

[0017] The present invention also provides a method for preparing trypsin, comprising the step of fermenting and culturing the above-mentioned recombinant host cells to prepare the trypsin.

[0018] The present invention also provides the application of the above-mentioned trypsin in the catalytic hydrolysis of proteins.

[0019] The present invention discloses the following technical effects:

[0020] This disclosure discloses the cloning of a trypsin gene, Try3, from a hot spring metagenomic library. The product obtained by expressing this gene in Pichia pastoris—recombinant Try3 trypsin—hydrolyzes Nα-benzoyl-L-arginine ethyl ester (BAEE). The trypsin activity of this recombinant Try3 trypsin is 734.2 U / mL under optimal conditions of pH 8.5 and 70°C. Enzymatic characterization studies revealed that this enzyme exhibits good tolerance within a pH range of 2.0-12.0, and retains over 70% of its activity after incubation at 60°C for 60 min. This enzyme demonstrates a wide pH tolerance range and good thermostability, well meeting the needs of industrial production. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 The PCR identification results of recombinant Pichia pastoris are shown in the figure; lane M is the Takara DL2000 Marker and lane P1 is the positive clone.

[0023] Figure 2 SDS-PAGE electrophoresis image of purified trypsin products; where lane M is the electrophoresis result of protein markers; lanes P1 and P2 are crude enzyme solution; lanes P3 and P4 are purified trypsin products;

[0024] Figure 3 Statistical graph of trypsin activity under different pH conditions;

[0025] Figure 4 Statistical graph of trypsin activity after standing at 25℃ for 1 hour under different pH conditions;

[0026] Figure 5 Statistical graph of trypsin activity under different temperature conditions;

[0027] Figure 6 Statistical graph of trypsin activity after incubation at different temperatures for 1 hour. Detailed Implementation

[0028] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0029] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0030] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0031] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0032] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0033] Terminology Explanation:

[0034] Metagenomics, also known as microbial environmental genomics or metagenomics, offers the advantage of bypassing traditional pure cultures by obtaining genetic information from all uncultured microorganisms in the environment. This allows for the analysis of microbial genetic information and the screening of functional genes. Through the analysis of metagenomic data and the screening of metagenomic libraries, a large number of uncultured microorganisms in extreme environments and their functional genes can be more easily discovered.

[0035] The term "protein" is intended to refer to a polymer of amino acid residues comprising at least nine amino acids linked by peptide bonds. The polymer can be linear, branched, or cyclic. The polymer can contain native amino acids and / or amino acid analogs, and it can be interrupted by non-amino acid residues.

[0036] The term "coding gene" refers to a segment of DNA sequence that ultimately directs protein synthesis through transcription and translation. These genes are the direct carriers of genetic information in an organism, expressing it as functional proteins.

[0037] The term "expression vector" is a man-made and modified DNA molecule (usually a plasmid or viral vector) whose core function is to introduce a foreign gene (usually a coding gene) into a specific host cell (such as bacteria, yeast, insect cells, or mammalian cells) and to guide the efficient and accurate transcription and translation of that gene, ultimately producing the target protein (usually a recombinant protein).

[0038] As used herein, "plasmid" refers to a reproducible DNA construct. Typically, plasmid vectors contain a selective marker gene that allows for the identification and / or selection of host cells carrying the plasmid as positive or negative in the presence of a compound corresponding to the selective marker. A variety of positive and negative selective marker genes are known in the art. By way of example, an antibiotic resistance gene can be used as a positive selective marker gene to select host cells in the presence of the corresponding antibiotic.

[0039] The term "host cell" is intended to refer to a cell containing the nucleic acid molecules of the present invention. A host cell can consist of a single type of cell or a group of different types of cells. A host cell can also be a heterozygous cell, that is, a cell resulting from the fusion of at least two different types of cells. A host cell can belong to a cultured cell line, a primary cell, a stem cell, or a proliferating cell. According to the present invention, the "host cell" is a recombinant host cell, i.e., a cell that contains exogenous genetic material. Therefore, the host cell is not a naturally occurring wild-type cell, but a molecular biology tool obtained through genetic manipulation techniques.

[0040] The "percentage (%) sequence identity" relative to a reference amino acid sequence refers to the percentage of amino acid residues in the candidate sequence that are identical to those in the reference amino acid sequence after sequence alignment and (if necessary) introducing gaps to obtain the maximum percentage sequence identity, without considering any conserved substitutions as part of the sequence identity. To determine the percentage of amino acid sequence identity, alignment can be performed in various ways within the art, such as using BLAST, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for sequence alignment, including any algorithms required to achieve maximum alignment across the full length of the compared sequences.

[0041] This disclosure also relates to amino acid sequence variants that can be prepared by introducing appropriate modifications into the nucleotide sequence of a coding molecule or by peptide synthesis. Such modifications include, for example, deletions, insertions, and / or substitutions of residues in the amino acid sequence of an antibody. Any combination of deletions, insertions, and substitutions can be performed to obtain a final construct with desired properties, such as antigen-binding activity. Sites used for substitution typically include HVRs and frames (FRs). Possible amino acid substitutions include, but are not limited to: conserved substitutions of Ala (A) including Val, Leu, and Ile, preferably Val; conserved substitutions of Arg (R) including Lys, Gln, and Asn, preferably Lys; conserved substitutions of Asn (N) including Gln, His, Asp, Lys, and Arg, preferably Gln; conserved substitutions of Asp (D) including Glu and Asn, preferably Glu; and conserved substitutions of Cys (C) including S... er, Ala, preferably conservatively substituted with Ser; conservative substitutions of Gln(Q) include Asn, Glu, preferably conservatively substituted with Asn; conservative substitutions of Glu(E) include Asp, Gln, preferably conservatively substituted with Asp; conservative substitutions of Gly(G) include Ala; conservative substitutions of His(H) include Asn, Gln, Lys, Arg, preferably conservatively substituted with Arg; conservative substitutions of Ile(I) include Leu, Val, Met, Ala, Phe, Nle, preferably Conservative substitution for Leu (L) includes Nle, Ile, Val, Met, Ala, and Phe, with Ile being the preferred conservative substitution; conservative substitution for Lys (K) includes Arg, Gln, and Asn, with Arg being the preferred conservative substitution; conservative substitution for Met (M) includes Leu, Phe, and Ile, with Leu being the preferred conservative substitution; conservative substitution for Phe (F) includes Trp, Leu, Val, Ile, Ala, and Tyr; Tyr; conservative substitution for Pro (P) includes... Conservative substitutions include Ala; conservative substitutions of Ser(S) include Thr; conservative substitutions of Thr(T) include Val and Ser, with Ser being the preferred conservative substitution; conservative substitutions of Trp(W) include Tyr and Phe, with Tyr being the preferred conservative substitution; conservative substitutions of Tyr(Y) include Trp, Phe, Thr, and Ser, with Phe being the preferred conservative substitution; conservative substitutions of Val(V) include Ile, Leu, Met, Phe, Ala, and Nle, with Leu being the preferred conservative substitution.

[0042] The term "enzyme activity" is intended to describe the efficiency of an enzyme in converting a substrate into a product under given conditions. Enzyme efficiency, as described herein, considers both the rate at which the enzyme converts the substrate into a product and the extent to which the enzyme converts the substrate into a product. The expression "the extent to which the enzyme converts the substrate into a product" is intended to represent, for a given amount of enzyme, the ratio between the amount of final product obtained and the initial amount of substrate.

[0043] Example 1: Bioinformatics Analysis of the Trypsin Gene

[0044] Analysis of the metagenomic data of microorganisms from hot springs at temperatures of 42-90℃ (from the JGI-IMG Microbial Functional Genome Database) revealed an 837bp gene, Try3, encoding trypsin, which was then sent to Shanghai Sangon Biotech Co., Ltd. to synthesize its gene sequence.

[0045] The nucleotide sequence of the gene Try3 is shown in SEQ ID NO.1, and the amino acid sequence of the trypsin it encodes is shown in SEQ ID NO.2.

[0046] SEQ ID NO.1:

[0047] caaatggatgatccaactcatgctagaccaactagaccagctttgtttccaactcaaccatctgatggtatgcaaccacaaattattggtggtaatccagctgatgatggtgaatacccatggcaagttgctttggttgattctacttacattaatccatttgatggtcaattttgtggtggttctattattgctccaggttgggttttgactgctgctcactgtgttgttgatgatgatggtactgtttctaacccagctactttggatgttgttgctggtgttaacttgctttcttctggtccaacttctggttctcaaggtcaaagaagaaaggttgctcaaattattgtttacccatctttttactatactaatggaaagattccagaaaacgatattgctttgttgagattggctgctccattgaacttgaaccaaaaggctcaaccaatttccttggctactcaagctgattctcctagatttgctccaggtgttactgctactgtttctggttggggtagaatgaatcctcctccacaagaacctattttgtttccagatgctttgatggaagttcaagtcccaattgttgatcaaactatttgttctgctgcttatcctggtgaaatcactcctaatatgttgtgtgctggttacgctgctggtggatacgattcttgtcaaggagattctggtggtccattgattgttcctgatggtaacggtggttggttgcaagctggaattgtttcttggggaaagggttgtgctcaaccaaacaagtacggtgtttacactagattggctaactacactgaatggattaactct。

[0048] SEQ ID NO.2:

[0049] QMDDPTHARPTRPALFPTQPSDGMQPQIIGGNPADDGEYPWQVALVDSTYINPFDGQFCGGSIIAPGWVLTAAHCVVDDDGTVSNPATLDVVAGVNLLSSGPTSGSQGQRRKVAQIIVYPSFYYTNGKIPENDIALLRLA APLNLNQKAQPISLATQADSPRFAPGVTATVSGWGRMNPPPQEPILFPDALMEVQVPIVDQTICSAAYPGEITPNMLCAGYAAGGYDSCQGDSGGPLIVPDGNGGWLQAGIVSWGKGCAQPNKYGVYTRLANYTEWINS.

[0050] Example 2: Pichia pastoris expression of trypsin gene Try3

[0051] 1. Construction of recombinant expression vector in host Pichia pastoris

[0052] The Try3 gene and the pPIC9 vector were ligated using homologous recombination. The forward primers used were GAAGAAGGGGTATCTCTCGAGAAAAG (SEQ ID NO.3) and CCTCGAGAAAAGAGAGGCTGAAGCTCAAATGGATGAT (SEQ ID NO.4), and the reverse primer was GTGGTGACTCGCCGGCGCTTAATTAAGCG (SEQ ID NO.5). The pPIC9 vector was digested with restriction endonucleases XhoⅠ and NotⅠ. The PCR product of Try3 and the linearized vector were recombined using the Novizan ClonExpress Ultra OneStep Cloning Kit V2 to obtain the recombinant plasmid pPIC9-Try3.

[0053] 2. Transformation and validation of recombinant plasmids in Escherichia coli

[0054] The recombinant plasmid pPIC9-Try3 constructed above was transformed into E. coli TOP10 competent cells. Plasmids were extracted from colonies grown on LB agar plates containing ampicillin.

[0055] 3. Transformation, expression, and purification of the expression product of the Try3 gene in Pichia pastoris.

[0056] The extracted plasmids were linearized with the restriction endonuclease SacⅠ and concentrated with isopropanol. Pichia pastoris GS115 was cultured to OD... 600The concentration was approximately 0.6, and the bacterial cells were washed with pre-cooled pure water and sorbitol respectively. The concentrated plasmid was then electroporated into the competent cells dissolved in sorbitol.

[0057] Genomic templates were extracted from colonies grown on MD plates, and PCR amplification was performed using primers SEQ ID NO.6 / SEQ ID NO.7 to identify positive clones. Results are as follows: Figure 1 The results showed that both the AOX gene naturally present in the yeast genome and the transformed sequence were amplified, proving that it was a positive clone.

[0058] SEQ ID NO.6: CTGGTTTCAATTGACAAGC;

[0059] SEQ ID NO.7: TGGCATTCTGACATCCTC.

[0060] Positive clones were selected and grown in BMGY medium until OD. 600 2. Take the bacterial culture into a 50 mL centrifuge tube, and resuspend and wash it with sterile physiological saline. Resuspend the yeast in BMMY medium until OD200. 600 The concentration was 1. Starting from the time of transfer to BMMY medium, methanol was added every 24 hours to a final concentration of 1% to induce the expression of exogenous proteins.

[0061] The supernatant obtained after 6 days of culture was the crude trypsin enzyme solution. The crude enzyme solution was purified using a Shanghai Sangon Biotech nickel column and eluted with 50 mM elution buffer. The elution buffer formulation was as follows: 50 mmol / L imidazole, 20 mmol / L Tris-HCl, 500 mmol / L NaCl, pH 8.

[0062] The results of crude enzyme solution and purified SDS-PAGE electrophoresis are as follows: Figure 2 Show. From Figure 2 The single band observed during elution with the elution buffer indicates that high-purity Try3 target protein can be obtained under these conditions. All eluents containing the target protein were combined and concentrated via dialysis using a 10 kDa ultrafiltration tube (Merck, Millipore, USA). Simultaneously, the buffer was replaced with 20 mM NaH₂PO₄ at pH 7.4 to remove imidazole and high concentrations of NaCl from the enzyme solution and to adjust the pH, yielding purified recombinant Try3 trypsin.

[0063] Example 3: Property Analysis of Recombinant Try3 Trypsin

[0064] 1. Assay of trypsin activity

[0065] The enzyme activity of the recombinant Try3 trypsin prepared in Example 2 was determined using the BAEE method, and the specific method is as follows:

[0066] In a 3.2 mL reaction system, 2.7 mL of phosphate buffer (a mixture of 13 mL of 0.067 mol / L potassium dihydrogen phosphate and 87 mL of 0.067 mol / L disodium hydrogen phosphate, pH 7.6), 0.3 mL of substrate stock solution (85.7 mg of Nα-benzoyl-L-arginine ethyl ester hydrochloride dissolved in water to a final volume of 100 mL) and 0.2 mL of enzyme solution were mixed. The absorbance at 253 nm was measured using a UV spectrophotometer (with an inactivated enzyme solution as a blank control). Absorbance values ​​were read every 30 seconds for a total of 5 minutes. A graph was plotted with absorbance on the ordinate and time on the abscissa; the absorbance change should remain constant between 0.015 and 0.018 every 30 seconds, and the linear change should last for at least 3 minutes. The enzyme activity units (P / mL) were calculated using a standard curve after subtracting the control from the sample measurement.

[0067] P = (A1 - A2) / 0.003 / T / W;

[0068] Where P is the amount of trypsin in 1 mL of enzyme solution, in U / mL; A1 is the absorbance value at which the line terminates; A2 is the absorbance value at the beginning of the line; T is the time from A1 to A2, in minutes; W is the volume of enzyme solution, in mL; enzyme activity unit (U) is defined as: 1 U is the amount of enzyme required to catalyze a 0.003 change in the absorbance value of the substrate solution per minute.

[0069] The results showed that the specific activity of the recombinant Try3 trypsin was 734.2 U / mL under optimal conditions of pH 8.5 and 70℃.

[0070] 3. Determination of optimal pH and pH stability

[0071] (1) Determination of optimal pH

[0072] The recombinant Try3 trypsin prepared in Example 2 was tested for enzyme activity under different pH conditions. The pH corresponding to the highest enzyme activity is the optimal pH of the enzyme.

[0073] The optimal pH for recombinant Try3 trypsin was determined within the range of 2-12, with each increment being 0.5. The buffer was Britton-Robinson buffer (buffer range: pH 2-12). The reaction was performed and the enzyme activity of trypsin was determined using the same method as described in "1. Determination of trypsin activity" above, except that the pH during the reaction was changed. The highest enzyme activity was defined as 100%, and the relative activity under other conditions was calculated (repeated three times).

[0074] Figure 3 The results showed that the optimal pH for this recombinant Try3 trypsin was 8.5.

[0075] (2) pH stability analysis

[0076] The recombinant Try3 trypsin prepared in Example 2 was incubated at 25°C for 1 hour at different pH values, and then the substrate was added for reaction. The enzyme activity of the trypsin was then measured at the optimal pH.

[0077] pH stability determination: The enzyme solution was incubated at 25°C for 1 hour at the corresponding pH, and then the enzyme solution was added to the substrate and reacted at the optimal pH. Other reaction conditions were the same as described in "1. Determination of trypsin activity". The enzyme activity of trypsin was determined according to the same method as described in "1. Determination of trypsin activity". The highest enzyme activity was defined as 100%, and the relative activity under other conditions was calculated (repeated three times).

[0078] Figure 4 The results showed that the recombinant Try3 trypsin has a wide pH tolerance range.

[0079] 4. Determination of optimal temperature and thermal stability

[0080] (1) Determination of optimal temperature

[0081] The recombinant Try3 trypsin prepared in Example 2 was tested for enzyme activity under different temperature conditions. The temperature at which the enzyme activity was highest was the optimal temperature for the enzyme.

[0082] The optimal temperature range for recombinant Try3 trypsin was set to 20-85℃, with each temperature gradient being 5℃. The optimal temperature was determined as follows: the substrate solution was incubated at different temperatures for 5 minutes, then the enzyme solution was added and quickly mixed. The mixture was then placed in the corresponding temperature for reaction, with other reaction conditions the same as described in "1. Determination of Trypsin Activity". The enzyme activity was then determined according to the same method as described in "1. Determination of Trypsin Activity".

[0083] Figure 5 The results showed that the optimal temperature for recombinant Try3 trypsin was 70℃.

[0084] (2) Temperature stability analysis

[0085] The recombinant Try3 trypsin prepared in Example 2 was incubated at different temperatures for 1 hour, and then the reaction substrate was added. The enzyme activity was then measured at the optimal temperature.

[0086] Temperature stability was determined at 30-65℃. The enzyme solution was incubated at different temperatures for 1 hour, and then the enzyme solution was added to the reaction substrate and reacted at the optimal temperature. Other reaction conditions were the same as described in "1. Determination of trypsin activity". The trypsin activity was then determined using the same method as described above. The highest enzyme activity was defined as 100%, and the relative activities under other conditions were calculated (repeated three times).

[0087] Figure 6 The results showed that the recombinant Try3 trypsin has good thermal stability.

[0088] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A trypsin, characterized in that, The trypsin is any one of (1)-(2): (1) Try3 protein, the amino acid sequence of which is shown in SEQ ID NO.2; (2) The fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of (1).

2. A gene encoding trypsin as described in claim 1.

3. The encoding gene according to claim 2, characterized in that, The nucleotide sequence of the encoding gene is shown in SEQ ID NO.

1.

4. A gene expression cassette, characterized in that, Includes the coding gene as described in claim 2 or 3.

5. A recombinant expression vector, characterized in that, Includes the gene expression cassette as described in claim 4.

6. A recombinant host cell, characterized in that, Includes the recombinant expression vector as described in claim 5.

7. The recombinant host cell according to claim 6, characterized in that, The recombinant host cell is a recombinant yeast.

8. The use of the encoding gene as described in claim 2 or 3, the gene expression cassette as described in claim 4, the recombinant expression vector as described in claim 5, or the recombinant host cell as described in claim 6 or 7 in the preparation of the trypsin as described in claim 1.

9. A method for preparing trypsin, characterized in that, The step includes fermenting and culturing the recombinant host cell as described in claim 6 or 7 to prepare the trypsin.

10. The use of the trypsin as described in claim 1 in the catalytic hydrolysis of proteins.

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